ES2857080T3 - New cigarette filter containing alginite - Google Patents

Abstract

The use of alginite for the filtration of cigarette smoke.

Description

DESCRIPTION

New cigarette filter containing alginite

Field of the invention

The present invention relates to a cigarette filter. In particular, the present invention relates to a new cigarette filter, in which materials of natural origin are used that have not been applied before in this special field. More particularly, the present invention relates to a cigarette filter, which can be used to adsorb the toxic components of cigarette smoke, and reduce the tissue damage that is triggered by cigarette smoke in the respiratory organs, the cardiovascular system and the mucosa. Especially the present invention relates to a cigarette filter containing alginite.

Technical background

Smoking is a pervasive, harmful human habit that is known to cause serious and often irreversible damage to health. Currently, smoking is one of the most highly documented etiological factors that contribute to the development of lung cancer and chronic obstructive pulmonary disease (COPD). The damage to health caused by smoking generates serious social and financial problems worldwide. For example, in the EU countries alone the premature death of more than 500,000 people is caused by the harmful effects of smoking.

About 50 years ago, the US Office of the Surgeon General published its first report on smoking and health (US Department of Health, Education, and Welfare, 1964). This report estimated that the average smoker had a 9-10-fold chance of developing lung cancer compared to a non-smoker, while heavy smokers had an approximately 20-fold increased risk. Furthermore, the report pointed out that smoking was the main cause of chronic bronchitis and that there was an association between smoking and emphysema as well as cardiovascular disease. It should be noted that chronic bronchitis and emphysema are currently considered as two aspects of chronic obstructive pulmonary disease (COPD). Over the past 50 years, the US Office of the Surgeon General has published numerous reports on smoking and health, some dealing with specialized topics such as smoking cessation, smoking during pregnancy, and second-hand smoke. The most recent report was published in 2014 - exactly 50 years after the first report (US Department of Health and Human Services, 2014).

In the last 50 years, the list of diseases associated with smoking has expanded considerably. Focusing only on cancer, there are currently numerous types of cancers that are associated with smoking in addition to lung cancer, including cancers of the upper respiratory tract (oropharynx, pharynx, trachea, and bronchi), stomach cancer, liver cancer, kidney cancer, pancreatic cancer, bladder cancer, cervical cancer, colorectal cancer, and acute myeloid leukemia. Additionally, the US Surgeon General indicates that perhaps as many as 20 million Americans have died prematurely over the past 50 years due to the effect of smoking. Given the obvious deleterious effects of smoking, mitigating these effects is a huge health concern, and any steps that can be taken to reduce the problem are clearly worth investigating. Without a doubt, the best action is to stop smoking. The benefits of quitting smoking are well known (see, for example, Fagerstrom, 2002). However, there are many smokers who either choose not to quit or who find it very difficult to quit. Although giving up smoking would be the most effective measure, the use of new technology, such as novel filters that effectively remove harmful constituents of smoke, could significantly reduce tobacco-related illnesses. As a consequence, any measure that can be taken to reduce the health effects of smoking will be of significant benefit. Without doubt, the most obvious attempt to mitigate the health effects of smoking through cigarette modification is through the addition of a cigarette filter. However, the use of filters has not been particularly successful.

One of the first proposals to add a filter to cigarettes was undoubtedly made by Ernst Wynder, an epidemiologist who was one of the first scientists to demonstrate the association of cigarette smoke with lung cancer. A first co-authored study by Wynder published in 1988 evaluated the difference in lung cancer risk between smokers of filtered cigarettes and smokers of unfiltered cigarettes (Wynder & Kabat, 1988). This study looked for the difference between these two types of smokers with respect to Kreyberg I (KI) and Kreyberg II (KII) cancers. (The Kreyberg nomenclature was in effect at the time, with KI lung cancers including squamous cell lung carcinoma, large cell lung cancer, and small cell lung cancer, while KII lung cancers comprise only adenocarcinoma. A reduction of approximately 45-50% was found for both men and women with respect to KI tumors, although none was statistically significant, while only a weaker difference was observed in men and no difference in the women for KII tumors. Cigarette filters became extremely popular during the second half of the 20th century, with approximately 0.5% of filter cigarettes sold in 1950 increasing to 88.5% in 1976 in the US (National Institute on Drug Abuse, 1977). Currently about 100% of the cigarettes sold worldwide are filter cigarettes. During the same period of time when the use of filters increased to a rapid In the US (1950-1976), sales-weighted cigarette tar supplies that are measured by machine decreased from 37 mg to 16 mg (Hoffmann D et al., 1996). The decline in tar supply throughout this period was a consequence of two trends. The first, as noted above, was simply a rapid increase in filter cigarette use. The second, however, was a consequence of the increased efficiency of the filters over time. A cigarette filter is conceptually quite simple, consisting of a porous plug of a given material that can absorb both the cigarette tar and the gas phase. Although some of the early filters used paper fibers as the absorbent material, today the vast majority of filters use cellulose acetate fibers. The filter, therefore, is simply a paper tube filled with cellulose acetate that is attached to the cigarette through the use of an envelope. An increase in efficiency can be achieved both by increasing the mass of cellulose acetate in the filter and by decreasing the diameter of the filament. Both approaches can only be taken so far, however, because eventually the draw resistance of the cigarette becomes large enough that the product is unacceptable to the consumer. The approach that was taken by virtually all tobacco companies to solve this problem was to introduce perforations in the filter casing. Therefore the smoker inhales a mixture of air and smoke. The vents reduce the draw resistance, and by taking in the air as well as the smoke, the smoke is diluted and the supply of smoke constituents is reduced. The greater the range of ventilation, the greater the amount of air and the less the amount of smoke that is inhaled by the smoker. Although most experts agree that a filtered cigarette reduces the risk of smoking by at least to a degree compared to an unfiltered cigarette, low-tar cigarettes, as they are called, reduce the supply of tar even less than could be achieved with a normal cigarette filter did not appear to lead to a health benefit. This conclusion was based on both population data and epidemiological studies. Considerable data were presented documenting the fact that smokers significantly compensate when they smoke a 'low tar cigarette' to either maintain nicotine level or flavor level, thereby increasing the actual supply of smoke by above the performance that is measured by the machine. In addition, a number of scientists expressed concern that the smoker could deliberately or inadvertently block the vents, thereby also significantly increasing the supply of smoke (US Department of Health and Human Services, 2001). A tangible result of these concerns is that cigarette packs are no longer allowed to display tar and nicotine yields that are machine-measured in at least the US and EU. Despite these problems, it is still possible to develop novel filters that can reduce the health effects of smoking, particularly if such filters can be developed without the need for filter ventilation. Such filters could be designed to selectively remove specific components of the semi-volatile smoke and gas phase of interest. It is important to note that smoke is made up of the gas phase, semi-volatile and the particle phase. The components for which there is evidence regarding health effects can be found in all three phases. There is no current technology that allows the selective filtration of components in the particle phase; however, both gas phase and semi-volatile components can be selectively filtered. An excellent example of such a filter currently in commercial use is the carbon filter. Virtually the entire Japanese market consists of carbon filter cigarettes, while about 50% of South Korean smokers use these products. Other technological advances have been made in filter development, but none of them are currently in significant commercial use. Currently the filter is a segment that is integrated directly into the cigarette at the end of the side of the mouth, so that the cigarette smoke must pass through the filter before entering the airways and lungs. Currently only 3% of all cigarettes in the world are sold without a filter. Although the amount of harmful substances reaching the smoker can be reduced by cigarette filters, this is generally achieved simply by reducing the amount of smoke reaching the mouth end of the cigarette. In most cases there is little or no selective filtration. Researchers are therefore highly interested in building a cigarette filter, which can selectively remove certain hazardous constituents from smoke to reduce the health consequences of smoking.

Cigarette smoke contains many reactive particles, such as low molecular weight carbonyl compounds, free radicals, quinones, hydrogen cyanide, nitrogen oxides, and aromatic amines, which are highly toxic, mutagenic, and carcinogenic. Therefore, selectively reducing the amount of these substances in cigarette smoke can reduce the health risks of smoking.

Increasingly, government regulations require higher filtration efficiency to reduce the amount of tobacco smoke supplied to the smoker. By using currently available cellulose acetate filters, some selectivity can be achieved by doping the filter with increasing concentrations of particles such as activated carbon or other natural substances. However, the increase in the concentration of particles changes the suction characteristics for smokers. In addition, the activated carbon particles in the filter help to reduce the amount of harmful volatile substances in cigarette smoke, but due to the lack of unpaired electrons, they cannot provide the most needed electron to supplement the unpaired electrons of the free radicals. Therefore, charcoal is not suitable for counteracting the impact of free radicals on various tissues, which contribute to inflammation and other harmful processes in the body that are triggered by cigarette smoke.

An important property of a cigarette is the encapsulated pressure drop. The term "encapsulated pressure drop" or "EPD" refers to the static pressure difference between the two ends of a cigarette when it is traversed by a flow of air under stable conditions. Higher EPD values mean that the smoker has to breathe into a device to smoke harder.

Because the increased efficiency of conventional filters increases the EPD of the filters, the public, and consequently manufacturers, have been slow to adopt these products. Therefore, there remains interest in developing improved and more effective filters that minimally affect the aspiration characteristics of cigarettes while removing the higher levels of certain constituents in conventional tobacco smoke, such as the constituents mentioned above. as well as carbon monoxide and phenols.

The most commonly used filler in the manufacture of cigarette filters is cellulose acetate which has a degree of substitution of approximately 2.5 acetate groups per anhydroglucose unit. During manufacture, the acetate polymer is typically extruded as a fiber tow and mixed with one or more plasticizers (eg, triacetin, polyethylene glycol, glycerin). Cellulose acetate tow processes are described, for example, in US Pat. No. 2,953,838 to Crawford et al. And US Pat. Number 2,794,239 to Crawford et al. Various fluids can be injected into the multifilament fiber tow used in the manufacture of tobacco smoke filters. These fluids, which can be used in tow alone or in combination with liquid or gaseous carriers, can be flavorings, tow blowing agents, lubricants, sizing solutions, finishing compositions, plasticizers or the like. Such fluids are intended to impart desired flavor or physical characteristics to cigarette smoke through the tow that is fluid treated. Fluid injection processes are set forth, for example, in US Patent. Rivers number 5,387,285.

The cellulose acetate fibers that typically form the filter element are coated with a fiber finishing composition. Such compositions are generally multi-component water-based emulsions. Each component can fulfill a specific function either during the processing of the fibers or during the subsequent use of a filter that is formed from the fibers. Typical components of a fiber finishing composition include lubricating oils to reduce friction so that the fibers can be processed without breaking, antistatic agents to reduce the build-up of static electricity in the fibers, and emulsifiers to inhibit phase separation in a formulation. fiber during processing. Other auxiliary components can include antimicrobial agents, hydrophilic agents, or other reactive compounds. After assembly of the fibrous tow into the ready-to-filter material, plasticizers can be applied to soften the fiber and allow inter-fiber bonds to form to harden the filter to a desired hardness / consistency. The surface chemistry of the cellulose acetate and plasticizer can provide a smoky flavor that is desired and widely accepted by smokers. Certain other filter designs / formulations may provide a different smoke flavor. To date, cellulose-free acetate tow filters have not been generally accepted and have not been commercially successful.

The state of the art contains several publications that relate to cigarette filters and several improvements that are applied here.

WO2013 / 1869838 describes a cigarette filter comprising a filter plug containing a cellulose ester staple fiber, a pulp and an alkali metal salt of a water soluble anionic polymer. The filter plug has an alkali metal content of 2 to 100 µmol per gram of the filter plug. The water soluble anionic polymer may comprise at least one member selected from the group consisting of a polyacrylic acid and a polysaccharide having a carboxyl group.

Japanese Patent Number 3677309 describes a cigarette filter material in the form of a sheet having a paper structure and comprising an uncurled cellulose ester staple fiber and a whipped pulp, wherein the whipped pulp has a degree of whipping Schopper-Riegler freedom of 20 to 90 ° SR, and the uncurled cellulose ester staple fiber is a staple fiber having an average fiber length of 1 to 10 mm and a fineness of 1 to 10 deniers. This document describes that a binder (for example, a water-soluble adhesive) can be used in the preparation of the sheet material, a binder as long as it does not have negative health effects, nor does it diminish the taste and palatability of the smoke from the tobacco, nor can it lead to disintegration of the filter material. In general, the amount of binder is preferably as small as possible (for example, no more than 10% by weight of the total weight of the material). An example in this document describes a sheet material that is formed from an uncurled cellulose acetate staple fiber and a pulp beaten by a wet paper production process, which is then sprayed with an aqueous solution of carboxymethylcellulose (3% in weight based on dry weight).

Japanese Patent Application Publication Number 7-75542 describes a cigarette filter comprising a tow of a cellulose ester fiber and a water soluble polymer which is contained in the tow and binds the fiber, the tow has been processed into a filter rod by using no more than 25 parts by weight of water relative to 100 parts by weight of tow. Examples in this document include a filter tip of cigarette that is obtained by adding 5 % by weight of a sodium salt of carboxymethylcellulose as a water-soluble polymer to an open cellulose acetate curly fiber tow and by feeding the openable tow into a machine wrapper for wrapping the tow that is opened with a filter wrap. Japanese Patent Application Publication Number 8-322539 (Patent Document 3, JP-8-322539A) describes a filter for cigarettes comprising a non-woven fabric consisting of a cellulose ester composition and a binder having a good dispersion in water, the non-woven fabric is wrapped in the form of a rod. Examples in this document include a filter plug that is produced by blowing a mesh wire with a cellulose acetate staple fiber through air flow for lamination or deposition, and by spraying the laminated material onto the wire with 10% by weight. of a 5% aqueous solution of a carboxymethylcellulose, by pressing and drying the wet laminate, the resulting nonwoven fabric is subjected to a crepe roll treatment and then the fabric is wrapped.

International Publication Number WO 2014/164492 refers to smoke filters that reduce the concentration of carbon monoxide and phenols in a smoke stream. Said filters include a porous mass section comprising a plurality of active particles, a plurality of binder particles and an active coating arranged on at least a portion of the active particles and the binder particles, wherein the active particles and the binder particles are join together at a plurality of contact points; and a filter section. In some cases, a filter may include a porous mass section comprising a plurality of active particles and a plurality of binder particles, wherein the active particles and the binder particles are bonded together at a plurality of contact points without an adhesive. ; and a filter section comprising an active dopant. Although this smoke filter may provide better results, it is quite complicated to prepare and the materials used to achieve the desired filtering effect are expensive.

A high efficiency cigarette filter is described in WO 2010/125412. The cigarette filter comprises, in addition to the common components of cigarette filters, pseudoboehmite (AlOOH.H2O), and components of grape, astaxanthin and blueberry as antioxidants. The advantageous effect of the cigarette filter is also due to the use of the grape components in the form of grape seeds and skin. US2003168071 describes zeolite and / or carbon absorbent minerals in a cigarette filter to filter tobacco smoke.

The document HU189383 describes a process to separate the gases that emanate from the decomposition of organic matter.

As mentioned above, it is well known that smoking is a major public health problem and a major etiological factor contributing to the development of lung cancer and chronic obstructive pulmonary disease. Therefore, the identification of new techniques to reduce cigarette-induced lung disease would be of considerable benefit.

Accordingly, the aim of the present invention is to provide a cigarette filter, which has the advantages of the solutions that already belong to the state of the art, but at the same time eliminates their drawbacks to the greatest extent possible. A further object of the present invention is to provide a cigarette filter that further reduces the harmful content of cigarette smoke compared to known cigarette filters.

Surprisingly, it was found that the objectives of the invention can be achieved successfully, if a natural substance, alginite, not used before for this purpose, is applied to the cigarette filter.

Our experiments showed that a significant reduction in the amount of harmful substances in cigarette smoke, compared to current filters, can be achieved by using alginite in the filters.

Detailed description of the invention

The present invention relates to a cigarette filter, which further reduces the harmful content of cigarette smoke compared to known cigarette filters. Said advantageous properties are due to the use of alginite in cigarette filters. Alginite can be used alone or in combination with other substances, already used in cigarette filters.

Alginite is a precipitated rock that consists of biomass of algae and tuff, volcanic dust disaggregated into clay. In the lakes of the Carpathian basin, intense volcanic activity occurred in the Pliocene about 3-5 million years ago. This activity created the well-known basalt mountains, at the same time forming special tuff rings. After the extinction of the volcanic activity, the tuff rings were flooded with water in this way the explosion lakes (maars) were formed. The water in the blast lakes was heated by hot springs, and the hot solutions they contained enriched the water with microelements, mineral salts and other nutrients. The elements in the mineral colloids that resulted from the degradation of the glass material of the volcanic tuff further enriched the nutritional content of the blast lakes. In the calm waters of the blast lakes accumulated large amounts of algae (especially the green algae Botriococcus braunii) and other floating animal or plant organisms. The plant and animal organisms that accumulated died and mixed with leaf debris and anther dust that washed off the dense shoreline vegetation and settled on the bottom of blast lakes. In the anoxic environment, along with the decaying tuff and other dead planktonic organisms, they accumulated as decaying sludge (sapropel). In the sedimentation phase of the blast lakes, larger animal bodies were introduced into the warm mud, and as a result, the mud was enriched with phosphorous materials. This depleted and hardened biomass underwent specific physical and chemical changes over several million years and took its current form: alginite rock.

Alginite is an earthy rock that has a clay structure consisting of lamellae that occasionally shed as leaves. Alginite has no toxic effect (see Dr. Solti Gabor: Az Alginit. Ismerteto tanulmány. Az Alginit a Mezogazdaságért es Kornyezetvédelemért Alapitvány tevékenysége (1993-2013) 2014). Its color is reseda (green) or gray that sometimes turns ocher. Their lamellar structure is best visible after drying and plant traces or plant debris can often be found between the lamellae.

Its most important physical property is that it can bind 0.5-1.01 water per kilogram. Alginite consists of 80-90% clay and silt fractions, and the deposition containing the coarsest particles near the shore. In the last phase of the sedimentation craters (lagoons) the content of organic matter decreased and the content of bentonite increased. The composition of alginite shows a large deviation in samples taken from the same places. The average humus content is 30%, occasionally reaching 45%. The average content of lime (in the form of CaCO3) is 33%, occasionally reaching 40%. Fossil biomass has been shown to contain 64 elements. This means that alginite is especially rich in macro and microelements, the most important elements being the following: nitrogen (N): 0.5%, phosphorus (in the form of P2O5): 0.6%, potassium (in the form of K2 O): 0.9%, magnesium (Mg): 1.0%. Typical mineral components are montmorillonite, illite, dolomite, calcite, aragonite, quartz gypsum, plagiarism class, siderite, magnesite, pyrite, and orthoclase. In addition to the above, the most important microelements are iron (Fe), manganese (Mn), copper (Cu), zinc (Zn), cobalt (Co), nickel (Ni), lithium (Li), titanium (Ti), chromium (Cr) and cadmium (Cd). One of the special characteristics of hummus ingredients is their biochemical effect to improve plant growth. When alginite is used in agriculture, humic acids exert an enzyme-like enhancing effect and also a hormonal effect and, through the regulation of the water absorption capacity of the roots, also an indirect enhancing effect on the plant growth.

Alginite finds widespread use for various purposes. In the cultivation of plants and fruits, alginite can be used for breeding. Its multiplied use increases soil fertility by 20-30% in the first year. Due to their clay minerals, artificial fertilizers must be used at a higher level, thereby increasing the transfer of phosphorus, nitrogen and potassium from the soil to groundwater, rivers and lakes. Its effect lasts 4-6 years. Alginite is a natural material, it retains its quality indefinitely, it cannot be abused and even higher levels of use have no adverse effects. Alginite can also be used as garden soil in the form of mixtures. Mixed with other natural materials such as zeolite, perlite, peat or basalt, highly efficient and agent-free soil mixtures have been prepared. The use of alginite results in an increase in the quantity and quality of yield in the cultivation of olive trees and ornamentals either in the garden or in polytunnels at harvest time. Alginite can also be used as a starter in the hole planting of forest tree species. The use of alginite results in a 6-13% quantitative increase and 20% faster growth. Alginite suspension spraying in autumn has a protective effect on plants and aids in the hibernation of trees, while spraying in spring provides protection against pests. As a result of alginite spraying, the manganese, iron, zinc and copper content of the plants increases, while the calcium content in the fruits provides more flavor and a longer shelf life. In livestock, alginite combined with liquid manure provides a highly effective product for use as a complementary treatment for organic fertilizers, or to replace them. Alginite reduces the degradation period of the fertilizer and can be combined with other nutrients. The mixture of alginite with litter results in a more substantial fertilizer and improves the growth of domestic animals and poultry. Alginite also exerts environmental protective effects. Due to its high adsorption affinity, it effectively binds animal stall odors and reduces the concentration of SO2 and NH3 in the airspace (see, for example, Hungarian Patent Number 189,383: "Process for binding of gases with unpleasant smell produced by dissolving organic materials and for production of organic manure with high efficiency ").

Alginite's uses for humans include its use as a mud for joint, rheumatic and sports problems, and it also has the advantage of being made into a rheumatism ointment. Alginite is also useful against varicose veins and psoriasis and can also be used for skin regeneration and general improvement of skin condition. Furthermore, alginite can also be used as a base for medical air fresheners.

Alginite can be found in Hungary and is commercially available from numerous Hungarian companies, for example from Gérce-Alginit Kft, (Gérce, Hungary).

Surprisingly, alginite has now been found to be effective in a new technical field. Our studies show that alginite is especially effective when used in cigarette filters alone or in combination with other known ingredients, as discussed below. Unexpectedly, it was found that the use of alginite in cigarette filters resulted in significantly less reactive oxygen species (ROS) from saliva, significantly less ROS formation from blood serum, less endothelial damage, less blood serum damage. lung epithelium, significantly higher glutathione level, less lung tissue damage and less inflammation in lung tissues, such advantageous properties are described in detail below.

The use of alginite causes significantly less reactive oxygen species (ROS) from saliva. Although saliva itself has a certain concentration of free radicals, cigarette smoke causes an increase in the level of free radicals. It is estimated that there are more than 1014 free radicals per puff of cigarette smoke (Church and Pryor, 1985; Church DF, Pryor WA, "Free-radical chemistry of cigarette smoke and its toxicological implications,", Environ Health Perspect, 1985, 64 : 111-26). Since free radicals can interact with numerous organic substrates to produce ROS, it is not surprising that cigarette smoke increases the ROS level in saliva. However, in addition to the radicals contained in cigarette smoke, significant radical formation, as well as the direct production of ROS, can arise from the inflammatory response elicited by cigarette smoke, leading to an increase in neutrophil and macrophage levels. (Messner and Bernhard, 2014; Messner B, Bernhard D, "Smoking and cardiovascular disease. Mechanisms of endothelial dysfunction and early atherogenesis," Arterioscler Thromb Vasc Biol, 2014, 34: 509-15). The antioxidant capacity of the untreated saliva of our volunteers was measured, who then smoked a cigarette, after which their saliva was collected again. The change in the level of antioxidant capacity of saliva was measured by using the smoke of a control cigarette. The same exercise was repeated with different filters containing both monoalginite alone and in four different combinations of alginite with - skin and grape seed (GSS), alginite - special Al oxide, alginite - zeolite alginite - carbon from the same filters in a mix 50-50%. All combinations of filters with alginite produced a significantly lower decrease in the antioxidant capacity of saliva compared to the control filter. Alginite alone produces a significant difference in antioxidant capacity compared to control, but all cigarettes combined fared significantly better than alginite alone, clear evidence that alginite and associated combinations act synergistically.

The use of an alginite filter caused significantly less ROS formation from blood serum. The experiment demonstrating this was similar to the saliva experiment, but was performed with blood serum. The serum itself has a certain concentration of free radicals. Although the serum itself has a certain concentration of free radicals, cigarette smoke causes an increase in the level of free radicals. It is estimated that there are more than 1014 free radicals per puff of cigarette smoke (Church and Pryor, 1985). Since free radicals can interact with numerous organic substrates to produce ROS, it is not surprising that cigarette smoke decreases the antioxidant capacity of serum. However, in addition to the radicals contained in cigarette smoke, significant radical formation, as well as direct ROS production, can arise from the inflammatory response elicited by cigarette smoke that leads to increased levels of neutrophils. and macrophages. (Messner and Bernhard, 2013). The antioxidant capacity of the untreated serum was measured. Our smoking machine was then used to channel all the cigarette smoke through an IV tube. The change in the antioxidant capacity of the serum was measured by using the smoke of a control cigarette. The same exercise was repeated with different filters containing both monoalginite alone and a combination of alginite with four different filter materials, i.e. alginite - grape skin and seed (GSS), alginite - special Al oxide, alginite - zeolite, alginite - carbon from the same filters. All the filters containing alginite produced a significantly less decrease in the antioxidant capacity of the serum compared to the control filter.

The use of alginite produced smoke that caused less endothelial damage. The cells that line the inner surface of the blood vessels are called endothelial cells. These cells play an important role in protecting these vessels. Once the endothelium is damaged, which is often called endothelial dysfunction, the risks of cardiovascular disease increase. Since smoke, when it leaves the lung through the alveoli, enters the bloodstream, exposure of the endothelium to smoke occurs and initially leads to endothelial dysfunction, well known to be a crucial first stage in the development of cardiovascular disease. that are related to smoking (Ambose and Barua, 2004; Ambrose JA, Barua RS, "The pathophysiology of cigarette and cardiovascular disease. An update," J Am Coll Cardiol, 2004, 43: 1731-7; Messner and Bernhard, 2014) . Endothelial cell damage that occurs when endothelial cells exposed to full smoke are compared to untreated cells was measured. Significantly less cell damage occurs when the same cell line is exposed to either alginite filtered smoke or alginite filtered smoke.

The use of alginite also resulted in smoke that caused less damage to the lung epithelium. The lung epithelium is the first line of defense against inhaled toxins. The alveolar epithelial cells of the lung are known to be damaged by exposure to smoke and even to cell death (Kosmider et al., 2011; Kosmider B, Messier EM, Chu HW, Mason RJ, "Human alveolar epithelial cell injury induced by cigarette smoke, "PLoS One, 2011, 6: e26059), which is evidenced by a decrease in the number of healthy cells compared to untreated cells. The filtered smoke containing alginite caused a significantly smaller decrease in the healthy cell count compared to a control cigarette. Since necrotic epithelial cells secrete proteins in the lung that trigger inflammation, which can eventually lead to lung cancer or COPD, they protect the epithelium by filters containing alginite and a combination of four different filter materials that pair with, ie, algite with - skin and grape seed), algite - special Al oxide, alginite - carbon from the same filters is clearly a health benefit of smokers.

Glutathione levels were also significantly higher with algine-filtered cigarette smoke compared to the control cigarette. Both epithelial and endothelial cell lines were exposed to control cigarettes and alginite and to a combination of alginite with - grapeseed skin (GSS), alginite - special Al oxide, alginite - carbon from the same filters, such as smoke-containing alginite. of filtered cigarette. The determination of glutathione levels indicated significantly higher levels of glutathione in the cells exposed to the smoke of the alginite filter cigarettes compared to the control cigarette. Since it is well known that glutathione protects against oxidative stress (Rahman and MacNee, 2000; Rahman I, MacNee W, "Oxidative stress and regulation of glutathione in lung inflammation," Eur Respir J, 2000, 16: 534-54) , this means that the filters containing alginite better protect the autochthonous defense mechanism of the lung against the damage of the lung tissue induced by oxidative stress than the control cigarette.

Alginite-filtered smoke caused less lung tissue damage and caused less inflammation compared to control cigarette smoke. A three-dimensional lung tissue, designated as spheroids, has been constructed from human cells with a known profile, specifically, lung epithelial cells, fibroblasts, endothelial cells, and macrophages. The three-dimensional construction allows cells to develop a functional organization, similar to that found in their counterparts in vivo. 3D models offer a much better experimental model to simulate the environment in vivo than conventional monoculture monolayer (2D) systems. The biochemical profile of a 3D tissue culture is strikingly similar to that of the living organism. 3D spheroids react to external stimuli in a manner similar to living peripheral lung tissue. Their inflammatory response is almost identical and they also produce surfactant. When these 3D spheroids were exposed to filtered cigarette smoke through new alginite cigarette filters, the level of the cytokines IL-8 and IL-6, which are known as inflammatory mediators, was expressed to a significantly lower degree in comparison. with control cigarettes.

As mentioned above, alginite can be used in the filters of the invention alone or in combination with other substances that are used in cigarette filters prior to the filing date of the present invention. Such materials, as well as their preparation and use, are known to those of skill in the art.

For example, when "carbon" or "grape" or "grape components" are mentioned with regard to cigarette filters, they refer to activated carbon and grapeseed and skin grated, although, from the prior art, it is apparent to one skilled in the art that grape components may also be present in other forms. These components, as well as their availability, are also well known to those of skill in the art.

The present invention is described herein in more detail through the following examples. The Examples are for illustrative purposes only. From the Examples, a person skilled in the art will readily understand that alginite even alone has significantly improved filter characteristics over known filter materials. Furthermore, the examples containing data on combinations containing alginite and certain filter materials belonging to the state of the art will make it clear to one of ordinary skill in the art that alginite acts synergistically with other filter materials. With regard to such materials we refer, for example, also to the free radical scavengers which are described in WO 2010/125412 mentioned above.

Example 1 .: The use of alginite causes a significantly lower increase in the antioxidant status of saliva and serum - Experiments of the Budapest University of Technology (BUT)

The aim of this study was to investigate the effects of different filters on the ability of cigarette smoke to alter the antioxidant status of samples (serum and saliva). Measurements of serum samples were carried out with the RANDOX® TAS assay. Serum samples were prepared by reconstitution of lyophilized serum, which was measured after reconstitution (blank) or after filtered cigarette smoke bubbled through. The total antioxidant status of saliva was measured before and after smoking conventional or experimental cigarettes equipped with filters according to the invention. The data acquired by our measurements could reflect the ROS and free radical binding capacity of the filters. Materials and procedures

Antioxidant status measurements with benzidine assay and the Randox® Total Antioxidant Status (TAS) Kit Measurements of antioxidant status were carried out using the widely accepted benzidine assay and the commercially available Randox® Total Antioxidant Status (TAS) kit. The benzidine assay uses a peroxide generation system (hydrogen peroxide and peroxidase) and a peroxide sensitive chromogen (benzidine). The peroxides generated in situ react with the chromogen to give an intermediate compound with an absorbance peak at 620 nm detectable with a spectrophotometer. The antioxidants present in the sample compete with the chromogen in its reactions with peroxides and make it difficult to generate the detectable signal. By comparing the detectable chromogen formation of the samples with a negative control with no antioxidants present and with a positive control with a known antioxidant concentration, the antioxidant status of the samples can be estimated.

Used reagents and instruments

Reagent A (dissolved in Type II purified water)

- Sodium chloride 155 mM (Reanal, cat. No. 24640-1-08-38)

- 25 mU / ml horseradish peroxidase (Sigma®, cat. No. 77332)

- 233 | ^j M benzidine dihydrochloride (Sigma®, cat. No. B3383)

Reagent B (dissolved in Type II purified water)

- ^250j M urea-hydrogen peroxide (Sigma®, cat No. 289,132.)

Samples

Saliva samples were taken from 17 subjects before and after smoking cigarettes. The volunteers were recruited by OF Laboratories at the Budapest University of Technology and Economics. Each volunteer reported between 8-9 am to collect saliva, smoke cigarettes, and collect saliva again. Each morning a test cigarette was smoked and saliva was collected. Each volunteer smoked 4 different cigarettes (distinguished by the filter); two between December 4-7, 2015 and two between January 5-8, 2016. Smokers were asked to report to smoke without having ingested any food or liquid that morning and not brushing their teeth. The saliva was cooled and taken for evaluation within the facilities of the BUT laboratories.

Serum samples were reconstituted from lyophilized serum (Analyticon Contronorm® PLUS), according to the manufacturer's instructions, in Type II purified water. Serum samples were measured directly (blank) or after filtered cigarette smoke was bubbled through the serum by OptiFilter. The cigarettes were smoked using the Filtrona SM302 8-port linear smoking machine. The cigarettes were smoked according to ISO 3308 with 100% of the filter vents blocked. The smoke was passed through a Cambridge filter (44mm glass fiber filter, Art.No .: 80202851, Borgwaldt KC), and the resulting gas phase was channeled through a silicone tube and bubbled into a container. flexible (shocking) containing 1.5 ml of serum solution. After each cigarette, the Cambridge filter pad was replaced with a new one, after each cigarette, the silicone tube was replaced with a new one. Filters were labeled 1-3.

Controls and Measurement Tools

- negative control (purified water type II)

- Positive Control (Randox® Total Antioxidant Status Kit Calibrator Standard, Cat. # NX 2332)

- Randox® Kit with total antioxidant status

- Spectrophotometer (Thermo Scientific ™ Multiskan ™ GO Microplate Spectrophotometer)

Benzidine Assay Procedure

Measurements were carried out with the microplate spectrophotometer described above, cells were incubated at 37 ° C in a 96-well plate. The reaction mix on the microplate was prepared as follows: 5 µl of sample or control and 250 µl of reagent A were pipetted into the wells. The mixture was then homogenized and then read by the microplate reader. An initial absorbance reading was determined at A = 620 nm prior to the addition of 50 µl of Reagent B to initiate peroxide generation, after which absorbance readings were determined at A = 620 nm from 0 to 3 minutes. . The absorbance results were considered the absorbance values that are measured at 2.5 minutes. All samples and controls were stored on ice and each sample was measured in 3 parallel wells for statistical analysis.

Randox® Total Antioxidant Status (TAS) Kit Procedure

Measurements were carried out according to the supplied manual using a microplate spectrophotometer described above. By using a microplate instead of a cuvette, all required reagent volumes were reduced by a factor of 4. This resulted in a final reaction volume of 305 | _il resulting from the addition of 5 | _il of sample or control, 250 | _il of Reagent A and 50 | _il of Reagent B as described in the manual.

Cigarette Material

The cigarettes that were used in the experiment were provided by OptiFilter Zrt. The specifications and manufacture of the cigarettes were as follows. The Kentucky 3R4F Reference Cigarettes were manufactured and assembled by the University of Kentucky, KY, USA. The reference cigarettes were provided to OptiFilter Zrt of Hungary by Celanese Corporation, Narrows, VA, USA. cigarettes were assembled, and the test cigarettes were produced by OptiFilter Zrt. CellFx filter rods were prepared and provided by Celanese Corporation. These contained different filter materials, sometimes mixed. Celanese Corporation manufactured and provided additional acetate filter materials with different textural characteristics, thus producing different pressure drop values. The 27 mm acetate portions of the Kentucky Reference Cigarette (KRC) 3R4F filter (2.9 / 41,000) were removed and discarded. The filter rods, which are manufactured using Celanese's CellFx technology, contained different filler materials. A selected filter rod was inserted that faces the burning surface of the cigarette, and an additional portion of acetate was selected and introduced into the filter, which ensures that the pressure drop value (total draw resistance) of the cigarette (filter ventilation closed) was the same as the KRC pressure drop value (aspiration resistance 170 mm H2O / -2%). The Celanese rods were 12mm long. The acetate parts were 15mm long. The total length of the filter was 27 mm.

Summary of the filters used in the experiment

Results

During the test which was carried out on saliva and serum, the following test results were obtained.

Serum experiment

Shows Capacity Status Alteration of status

ERO antioxidant antioxidant

Serum 1 Blank 33% 67%

Filter 1 44% 56% -16%

Filter 2 47% 53% -21%

Serum 2 Blank 21% 79%

Filter 3 30% 70% -11%

The alteration in the antioxidant status is shown in Figure 1.

Measurements were carried out in five repetitions. The results indicate that filter 3 is superior to the control cigarette (filter 1).

Results of Measurement of Saliva Samples with the Benzidine Assay

The measurement was carried out with 17 subjects. Each sample that was collected from the subjects was measured 3 times.

Average decrease in antioxidant status

Filter 1 Decrease in antioxidant status 29%

Filter 2 Decrease in antioxidant status 33%

Filter 3 Decrease in antioxidant status 12%

Statistical analysis of the experiment with saliva.

- The evaluation of the statistical significance of the changes in antioxidant status before and after cigarette smoking was carried out using the Wilcoxon combined pairs test (StatSoft-STATISTICA10). The results were considered significant for p <0.05.

The change in the antioxidant status of Filter 1 before and after smoking is significant. The results obtained from the test indicate a strong statistical difference.

The change in the antioxidant status of Filter 2 before and after smoking is significant. The results obtained from the test indicate a strong statistical difference.

There is no statistically significant change in the antioxidant status of Filter 3 before and after smoking; Although there is an observable difference (p = 0.065), it does not reach the threshold of statistical significance.

- The evaluation of the statistical significance of the changes in antioxidant status caused by smoking different cigarettes was carried out using the Wilcoxon combined pairs test (StatSoft -STATISTICA10). The results were considered significant for p <0.05.

The change in antioxidant status between filter 1 and filter 2 is not significant.

The change in antioxidant status between filter 1 and filter 3 is significant.

Representation of the results of the decrease in antioxidant status using a box plot diagram

The related boxplot plot of multiple variables is shown in Figure 2. The peripheral data points are shown separately (StatSoft - STATISTICA10).

Conclusions

Our results show that cigarette smoke that passed through either Filter 1 or 2 reduced the antioxidant status of the serum by 15-20%. Filter 3 resulted in a significantly lower decrease in antioxidant capacity, compared to the control.

The absorbance readings of the saliva samples were compared to positive and negative controls to assess antioxidant status. Our results indicate that cigarette smoke that passed through Filter 1 and 2 reduced the antioxidant status of saliva by approximately 30%, which was statistically significant, while Filter 3 showed a statistically 12% decrease. significant compared to the control cigarette. These results are consistent with those of the serum measurements. Our results show that the components of the filters of the invention have a significant effect on the ability of cigarette smoke to change the antioxidant status of the samples under the test conditions described.

Example 2 .: The use of alginite causes a significantly lower increase in the antioxidant status of saliva and serum - experiments by Prof. Tibor Szarvas

The effect on saliva and serum of cigarette smoke that is filtered through the filters of the invention was also tested in the following additional experiment.

Materials and methods

The cigarettes used in the experiment were the Kentucky 3R4F Reference Cigarettes, which were manufactured and assembled by the University of Kentucky, KY, USA The test cigarettes were provided to OptiFilter Zrt of Hungary by Celanese Corporation, Narrows , VA, USA The cigarette filters were assembled, and the test cigarettes were produced by OptiFilter Zrt. CellFx filter rods were prepared and provided by Celanese Corporation. These contained different filter materials, sometimes mixed. Celanese Corporation manufactured and provided additional acetate filter materials with different textural characteristics, thus producing different pressure drop values. The 27 mm acetate portions of the Kentucky Reference Cigarette (KRC) 3R4F filter (2.9 / 41,000) were removed and discarded. The filter rods, which are manufactured using Celanese's CellFx technology, contained different filler materials. A selected filter rod facing the burning surface of the cigarette was introduced, and an additional acetate portion was selected and introduced into the filter, which ensures that the pressure drop value (total draw resistance) of the cigarette (filter ventilation closed) was the same as the KRC pressure drop value (aspiration resistance 170 mm H2O / -2%). The Celanese rods were either 10mm or 12mm or 15mm long. The acetate parts were either 17mm or 15mm or 12mm long. The total length of the filter was 27 mm. Cigarettes that were fitted with CellFx filter rods containing different fillers were measured and compared to the control in this biological evaluation. The following filters were used in the experiments:

Experimental setup

Cigarettes were smoked in the OF laboratory of the University of Technology and Economics, Budapest, in a linear smoking machine, Filtrona SM302 8-port according to the ISO 3308 protocol. Cigarettes were smoked with the filter vents blocked . The cigarette smoke was passed through a Cambridge filter (44mm fiberglass filter, Art.no .: 80202851, Borgwaldt KC), and the resulting gas phase was channeled through a silicone tube and bubbled into a glass container (shocking) containing 1.5 ml of serum solution. After each cigarette, the Cambridge filter pad was replaced with a new one, and after each cigarette, the silicone tube was replaced with a new one.

Measurement of the antioxidant capacity of serum

For the evaluation of the free radical binding capacity of the new experimental cigarette filters, two methods were used:

1.) Randox - Total Antioxidant Kit (available from Randox Lab. Ltd., Crumlin, UK)

2.) HRP-Benzidine Peroxide Assay

The Contronorm Plus control serum was supplied by Analyticon Biotechnologies AG, Germany. Smoking cigarettes and treating the serum with smoke was performed in the OF laboratories at the University of Technology and Economics, Budapest, and Dr. Szarvas performed the reading tests at the Campus of the Central Research Institute for Physics, at the Center for Energy of the Hungarian Academy of Sciences, Budapest. Freshly prepared reagents were used. The control serum was dissolved in 5 ml of redistilled water. After the cigarette smoke (1 cigarette) passed through a Cambridge filter, the resulting gas phase was bubbled into 1.5 ml of dissolved serum according to ISO 3308 protocol with the filter vents blocked. Then, 20 µl of treated serum was mixed with 1 ml of Reagent 1 (composition provided below), homogenized and the reaction started with 200 µl of Reagent 2 (composition provided below). The change in absorbance was measured after 3 minutes. The absorbance of the bubbling serum was compared to the absorbance of the unreacted control serum. A blank value without control serum was determined using 20 µl double distilled water. Measurements were also carried out on a plate reader (parameters: 5 | _il of serum, 250 | _il of reagent R1, 50 | _il of reagent R2).

Randox Assay to Determine Total Antioxidant Status in Serum

Assay principle: ABTS (2,2, '- Azino-bis (3-ethylbenzthiazoline-6-sulfonate) is incubated with a peroxidase (metmyoglobin) and H2O2 to produce the radical cation ABTS +. This has a relatively stable blue-green color. , which is measured at 600 nm The antioxidants in the added sample cause the suppression of this color, in a degree proportional to its concentration.

Sample: Contronorm control serum.

Procedure

Wavelength: 600 nm

Cuvette: 1 cm light path

Temperature: 37 ° C

Measurement: against the air

Double distilled water is mixed with 1 ml of reagent R2. The standard is mixed with 1 ml of reagent R2. The sample is mixed with 1 ml of reagent R2. Each solution is mixed well, incubated to reach the required temperature, and the initial absorbance (A1) is read. 200 | _il of R3 are added to each solution. Mixing and the timer start simultaneously. The absorbance is read after exactly 3 minutes (A2). The total antioxidant status that is expressed in% is established by the comparison of the reagent-value of the serum.

HRP-Benzidine Peroxide Assay

Reagent 1: HRP (horseradish peroxidase) 9000 U / L, benzidine hydrochloride 233 pmol / L, sodium chloride 155 mmol / L,

Reagent 2: Carbamide peroxide 0.36 mmol / L

Solvent: Double distilled water

Instrument: UV-VIS spectrophotometer, temperature 25 ° C

Freshly prepared reagents were used. The control serum was dissolved in 5 ml of redistilled water. After filtering the cigarette smoke using a Cambridge filter (1 cigarette), the resulting gas phase was bubbled into an amount of 1.5 ml of serum solution according to the ISO 3308 protocol. filter were blocked. Then 20 µl of treated serum solution was mixed with 1 ml of Reagent 1 and homogenized, and the reaction was started with 200 µl of Reagent 2. The change in absorbance at 620 nm was measured immediately after 3 minutes. The absorbance of the bubbling serum was compared to the absorbance of the unreacted control serum. The blank result was obtained without control serum by using 20 µl of double distilled water. The results of the experiments are summarized in the Tables. Measurements were also carried out on a plate reader (parameters: 5 | _il of serum, 250 | _il of reagent R1, 50 | _il of reagent R2).

Results

1. HRP-Benzidine Peroxide Assay

Abbreviations Production Capacity Improvement in Type radicals O antioxidant from comparison with reagents ^(%) reagent ^(%) control ^(%) Kentucky Reference Cigarettes * RC 64.7 35.3

Celartse mono carbon rod (length 10 rtvn) Ce ríod-'O-C Sfc r <A ¿’j

Celanse Alginite-Carbon rod (length 10 mm) Ce Ron- '0-AC. 37.3 62.7 73 Cavity: 200 mg, Alginite-Grape (120 - 80 mg) Cuv 200- AG 33.2 61.8

Cavity: AJ-O - Grape (33 - 66 mg) Cav-fOO-AIOG 59.1 40.9 16

Celanse Alginite-Grape Rod (length 15 mm) tí »R o d -'5 -A or j 64.3 34 Cavity: Alginite-Grape

_{(75-75 mg)} Cav-1bO-AG> 44.7 55.3 ¿7

The antioxidant capacity and the improvement compared to the control are shown in Figure 3 and Figure 4 respectively.

2, comparison of the cavity filters and CelFx of the invention in serum

HRP-Benzidine Peroxide Assay

Type Abbreviations Production Capacity Improvement in radicals O antioxidant from comparison with reagents (%) reagent (%) control (%) Kentucky Reference Cigarettes KRC 76 24

^{Cavity: 160 mg, AI-0 GRAPE ALGINITE Cav-160-}

_{+ ZEOLITE CARBONO AIOGAZC} 00 40 64

Cavity: 160 mg, 50% AJ-0 and 50% Grape Cav-160-AIOG 65 36 47 Cavity: 150 mg, 50% Zeolite and 50% Grape Cav-150-ZG 7-1 26 7 Cavity: 150 mg, 50 % Alginite and 50% Grape Cav-150-AG 45 55 129 Celanse Rod of 15 mm: 50% Alginite and 50% Grape CelRod-15-AG ₆₉ 31 27 Celanse Rod of 15 mm: 50% Alginite and 50% CelRod-15 -AC 45 55 126 Carbon

The antioxidant capacity and the improvement compared to the control are shown in Figure 5 and Figure 6 respectively.

3, Randox trial

The serum experiment was repeated with the Randox antioxidant kit methodology. The results are shown below.

Abbreviated Samples Absorber- _Capacity ^{Production Decrease in Improvement in radical banking or} antioxidant ^{capacity compared with reagents (%)} of the antioxidant reagent the control (%) of the serum (%)

(%)

Reagent (Randox) 0.256 100

Calibration Standard 0.005 1.9 ^98.1

Serum Serum 0.136 53.1 46.9

Kentucky Reference Cigarettes KRC 0.151 58.9 _{41.1 12}

12 mm Celanse rod: 50% CelRod- _{0.123 48.04 51.96 -11 187} Alginite and 50% Grape 12-AG

12 mm ^{Celanse rod} _{: Carbon CelRod- 12-C} 0.154 60.1 39.9 15 -21 Cavity 200 mg: Alginite-Grape (120- Cav-200- _{80 mg) 0.145 53.9 46.1 2 86}

_AG

The antioxidant capacity, the change of antioxidant capacity to control the serum and the change of antioxidant capacity for the Kentucky Cigarette are shown in Figure 7, Figure 8 and Figure 9 respectively. The results of the serum with the Randox methodology confirmed that the filters of the invention, both in CellFx structures and in the cavity, significantly improve the antioxidant status that is triggered by cigarette smoke in the gas phase. Considering that cigarette smoke enters the bloodstream seconds after inhalation, the use of the filter of the invention can result in a healthier endothelium condition in smokers.

Measurement of the antioxidant capacity of saliva

In order to evaluate the change of free radical status of the saliva after smoking and to compare different filter cigarettes, the changes that are triggered in the smoker's saliva were measured and compared.

Materials and methods

The cigarettes used in the experiment were the Kentucky 3R4F Reference Cigarettes, which were manufactured and assembled by the University of Kentucky, KY, USA. The test cigarettes were supplied to OptiFilter Zrt of Hungary by Celanese Corporation, Narrows, VA, USA Cigarette filters were assembled, and test cigarettes were produced by OptiFilter Zrt. CellFx filter rods were prepared and provided by Celanese Corporation. These contained different filter materials, sometimes mixed. Celanese Corporation manufactured and provided additional acetate filter materials with different textural characteristics, thus producing different pressure drop values. The 27mm acetate portions of the Kentucky Reference Cigarette (KRC) 3R4F (2.9 / 41,000) filter were removed and discarded. The filter rods, which are manufactured using Celanese's CellFx technology, contained different filler materials. A selected filter rod facing the burning surface of the cigarette was introduced, and an additional acetate portion was selected and introduced into the filter, which ensures that the pressure drop value (total draw resistance) of the cigarette (filter ventilation closed) was the same as the KRC pressure drop value (aspiration resistance 170 mm H2O / -2%). The Celanese rods were either 10mm or 12mm or 15mm long. The acetate parts were either 17mm or 15mm or 12mm long. The total length of the filter was 27 mm. Cigarettes that were fitted with CellFx filter rods containing different fillers were measured and compared to the control in this biological evaluation. Experimental setup

Saliva samples were taken from 38 subjects before and after smoking cigarettes. The volunteers were recruited by the OF laboratories at the Budapest University of Technology and Economics. Each volunteer reported between 8 a.m. and 9 a.m. to collect saliva, smoke cigarettes, and collect saliva again. Each morning a test cigarette was smoked and saliva collected from it. Each volunteer smoked 6 different cigarettes (differentiated by filter) between October 19 - November 20, 2015. Smokers were asked to report to smoke without ingesting any food or liquid that morning and without brushing their teeth. The saliva was cooled and taken to the ^k F ^k I laboratories for evaluation.

HRP-Benzidine Peroxide Assay

Reagent 1: HRP (horseradish peroxidase) 9000 U / L, benzidine hydrochloride 233 | _imol / L, sodium chloride 155 mmol / L,

Reagent 2: Carbamide peroxide 0.36 mmol / L

Solvent: Double distilled water

Instrument: UV-VIS spectrophotometer, temperature 25 ° C

Freshly prepared reagents were used. The control was dissolved in 5 ml of redistilled water. Saliva that was collected from the volunteers was collected. Then 20 µl of treated saliva solution was mixed with 1 ml of Reagent 1 and homogenized, and the reaction was started with 200 µl of Reagent 2. The change in absorbance at 620 nm was measured immediately after 3 minutes. The absorbance of saliva that was collected after smoking was compared to the absorbance of unreacted control saliva. The blank result was obtained without control saliva by using 20 µl of double distilled water. The results of the experiments are summarized in the Tables.

The study involved 38 volunteers according to the following:

Results

The results of the test are shown in Figure 10 and in Figure 11 where in Figure 11. Cigarette 1 = Kentucky Ref, Cigarette 2 = Carbon Rod, Cigarette 3 = Alg-Grape Rod, and Cigarette 4 = Alg-Cavity. Grape Summary of the filters used in the experiment

Saliva Filter description Abbreviation

Cigarette Reference Control

Filter 1 Kent. I laughed.

from Kentucky

Filter 2 Celanse Rod 12 mm: Carbon CelRod-12-C

Filter 3 Celanse Rod 12 mm: 50%

CelRod-12-AG

Alginite and 50% Grape

Filter 4 Cavity Alginite Grape 200 mg 50- Cav-200-AG

fifty%

Results

Change in antioxidant capacity

CELROD- CELROD- CAV-200-

KRC

12-C 12-AG AG

41.7 42.2 16.9 11.9

conclusion

Our experiments with serum confirmed that the filters of the invention, both in CelIFx structures and in the cavity, significantly improve the antioxidant capacity that is triggered by cigarette smoke. Considering that cigarette smoke enters the bloodstream seconds after inhalation, we believe that these data suggest that the filters of the present invention may contribute to a healthier endothelium condition in smokers. Our experiments with saliva confirmed that the filters of the invention, both in CellFx structures and in the cavity, significantly improve the antioxidant capacity in the mouth. We believe this can contribute to a healthier mucosa in smokers.

Example 3 .: Effect of cigarette filter composition on smoke-induced death of endothelial and epithelial cells.

Cigarette smoke is a complex chemical combination characterized by high levels of oxidants. Increasing numbers of articles show that cigarette smoke induces the activation of pulmonary vascular endothelial cells, which is associated with the loss of endothelial barrier function. This loss is a hallmark of endothelial dysfunction. In this process the oxidative stress induced by cigarette smoke leads to endothelial cell damage, allowing the penetration of activated monocytes and macrophages. Damage to the endothelial barrier may even be an early element of lung injury in response to exposure to cigarette smoke.

Cigarette smoke has also been shown to induce apoptosis of lung alveolar tissue through apoptosis of its epithelial cells, which contributes to the development of chronic lung diseases such as emphysema. Although all cell types within the lung can be damaged by oxidative damage, epithelial cells are the primary target of oxidative injury as they are the first line of defense in the lung. Therefore, it is not surprising that cigarette smoke injury to the epithelium is an important process in the pathogenesis of lung diseases associated with smoking.

Numerous studies have shown that highly reactive components of smoke, volatile carcinogens, and reactive oxygen species (ROS) that are derived from cigarette smoke and cells that are damaged by cigarette smoke contribute to lung injury that involves epithelial injury through cell death and additional ROS production in activated epithelial cells. Therefore, the protection of the epithelium from injury by cigarette smoke is considered essential for the treatment of many lung diseases that are associated with cigarette smoking. Our investigations demonstrated that the composition of cigarette filters may be important in modifying the effect of cigarette smoke on induced death of epithelial cells, which represent the cell line's first encounter with cigarette smoke, as well as the damage to endothelial cells. Filters that could more effectively remove components of cigarette smoke that have the highest potential for damage to epithelial cells, as well as endothelial cells, could reduce cigarette smoke-induced lung damage.

Materials, Subjects and Methods

The cigarettes used in the experiment were the Kentucky 3R4F Reference Cigarettes, which were manufactured and assembled by the University of Kentucky, KY, USA The cigarettes were provided to OptiFilter Zrt of Hungary by Celanese Corporation, Narrows, VA , USA Cigarette filters were assembled and test cigarettes were produced by OptiFilter Zrt., CellFx filter rods were prepared and provided by Celanese Corporation. These contained different filter materials, sometimes mixed. Celanese Corporation manufactured and provided additional acetate filter materials with different textural characteristics, thus producing different pressure drop values. The 27 mm acetate portions of the Kentucky Reference Cigarette (KRC) 3R4F filter (2.9 / 41,000) were removed and discarded. The filter rods, which are manufactured using Celanese's CellFx technology, contained different filler materials. A selected filter rod was inserted that faces the burning surface of the cigarette, and an additional portion of acetate was selected and introduced into the filter, which ensures that the pressure drop value (total draw resistance) of the cigarette (filter ventilation closed) was the same as the KRC pressure drop value (aspiration resistance 170 mm H2O / -2%). The Celanese rods were either 10mm or 12mm or 15mm long. The acetate parts were either 17mm or 15mm or 12mm long. The total length of the filter was 27 mm. Cigarettes that were fitted with CellFx filter rods containing different fillers were measured and compared to a control in this biological evaluation.

Endothelial cells play a fundamental role in the development of COPD, since the barrier function of endothelial cells is essential for healthy lung function; therefore, loss of endothelial barrier function may contribute to leukocyte infiltration, a characteristic sign of lung diseases including COPD. Smoke-induced cell death and inflammation in endothelial cells contribute to the development of COPD. Here we show that by using different cigarette filter compositions we can modify the composition of the smoke and can mitigate harmful biological effects. Figure 2 shows that the smoke from filters containing the Alginite / Zeolite / Carbon / Grape mixture is less damaging to endothelial cells.

Epithelial cells are important components of lung tissue and play a significant role in the development of lung cancer and COPD. By using the A549 lung epithelial cell line, we demonstrated that filters containing the Alginite / Zeolite / Carbon / Grape mixture significantly reduce epithelial cell death, thus possibly leading to a decreased risk of COPD. The results showed that the filters of the invention containing the mixture of Alginite / Zeolite / Carbon and Grape subtract some components of the smoke and, therefore, cause less damage to the epithelial and endothelial cells of the lung.

Protection of epithelial and endothelial cells may contribute to the attenuation of cigarette smoke-induced COPD and the development of other respiratory diseases.

Preparation of cigarette smoke extract.

Preparation of the cigarette smoke extract was performed as previously described (Chen et al; Chen ZH, Lam HC, Jin Y, Kim HP, Cao J, Lee SJ, Ifedigbo E, Parameswaran H, Ryter SW, Choi AM. Autophagy protein microtubule-associated protein 1 light chain-3B (LC3B) activates extrinsic apoptosis during cigarette smoke-induced emphysema. Proc Natl Acad Sci USA 2010 Nov 2; 107 (44): 18880-5). For the preparation of the cigarette smoke extract, Kentucky 3R4F research reference filter cigarettes (Tobacco Research Institute, University of Kentucky, Lexington, KY) were smoked by using a peristaltic pump (VWR International) by using of the different types of filters. All smoke was collected. Each cigarette was smoked within 4 minutes with a remaining 15mm butt and bubbled through 7.5 ml of cell growth medium via a silicone tube. This solution, which is considered to be 100% strength cigarette smoke extract, was adjusted to a pH of 7.45 and used within 15 minutes after preparation. After smoking each cigarette the silicone tube was replaced with a new one.

HUVEC and A549 cell cultures and treatments.

HUVEC cells (Human Umbilical Vein Endothelial Cells) were obtained from Lonza (Anaheim, CA, USA) cat number: C2519A, and cultured in endothelial growth medium (Lonza, Anaheim, CA, EE .UU.) In a humid atmosphere containing 5% CO2. For cell death analyzes, 5 x 10 3 / well HUVEC was seeded per well in 96-well plates in endothelial growth medium containing growth factors and 2% serum. Before each experiment, the medium was replaced with fresh medium containing no growth factor and 1% serum and incubated with 10% smoke extract for 24 hours.

The A549 human alveolar adenocarcinoma basal epithelial cells were obtained from the European Collection of Authenticated Cell Cultures (ECACC) (cell line: A549, cat number: 86012804). A549 cells were grown in DMEM medium containing 10% FCS in a humid atmosphere containing 5% CO2. For cell death analyzes, A5495 x 103 cells / well were seeded in 96-well plates in DMEM medium containing 10% FCS and treated with 10% CS extract for 24 hours.

Cell Viability Assays

MTT Assay

Cells were seeded in 96-well plates at an initial density as provided in the Figures and cultured overnight prior to smoke treatment. After the incubation period, the media was removed and replaced for 4 h with RPMI containing an adequate amount of the MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide ) (Chemicon Inc., El Segundo, CA) (14). The MTT reaction was terminated by adding HCl to the medium to a final concentration of 10 mM. The amount of water-insoluble formazan blue dye that forms from MTT was proportional to the number of living cells and was determined with an Anthos Labtech 200 enzyme-linked immunosorbent assay reader at 550 nm wavelength after dissolving the blue precipitate. formazan in 10% SDS. All experiments were performed in at least 6 replicates and repeated three times.

Sulphorhodamine B (SRB) Assay.

Cells were incubated in 96-well plates for 24 hours as described above. The culture medium was then discarded and the cells were fixed in situ by adding 100 µl of cold 10% (w / v) trichloroacetic acid and incubated for 30 min at 4 ° C. The supernatant was discarded, and the plates were washed five times with tap water and air dried for 24 hours. A solution of SRB (100 | _il) 0.4% (w / v) in 1% acetic acid was added, and the plates were incubated for 20 min at room temperature. After staining, the unbound dye was removed by washing five times with 1% acetic acid, and the plates were air dried. The dye that bound was subsequently solubilized with 10 mM (200 | _il) Tris (pH 10.5), and the absorbance was read on a 96-well plate reader at 560 nm by subtracting the background measurement at 600 nm by means of the use of a Promega Glomax multimode detection system.

Results: effect of smoke on lung epithelium and human endothelial cells

As indicated, pulmonary epithelial cells play a fundamental role in the development of chronic obstructive pulmonary disease (COPD). Changes in the composition of the cigarette filter could potentially produce smoke that would reduce epithelial cell death compared to conventional cigarette smoke. Therefore, we analyze the role of different filter compositions in smoke-induced epithelial cell death. Figure 1 shows the effects of different filter compositions on cell death in 549 cells. The results shown in Figure 1 were obtained by using 10% smoke extract applied to cell cultures. However, the results using a 10% smoke concentration are likely to be more reasonable because increasing the smoke concentration showed the proliferative effect of cigarette smoke. The data in Figure 12 indicate that three filters of the invention containing alginite / zeolite / grape skin and seed grinding (GSSG), alginite / GSSG, and alginite / charcoal filters significantly reduce smoke-induced death of epithelial cells. A549.

Figure 13 shows the effects of different filter compositions on the smoke-induced cell death of human main umbilical vein endothelial cells (HUVEC). Here we use 4 cigarettes for each measurement for each filter and perform 6 replicates, and this data shows that filters of the invention containing various filter materials in the filter significantly reduce smoke-induced endothelial cell death. These experiments showed that the cigarette filters of the invention significantly change the survival pattern of both the epithelial and endothelial tissues that are exposed to cigarette smoke. This can be helpful in fighting respiratory and cardiovascular diseases that are triggered by cigarette smoke.

Example 4 .: Production of Inflammatory Cytokines Following Exposure to Cigarette Smoke in a Human 3D Lung Tissue Model

Cigarette smoke is a major factor associated with many complex lung diseases. Exposure to smoke can induce inflammatory responses through the release of inflammatory cytokines. Macrophages play an important role in the inflammatory response and are particular sources of interleukin-8 (IL-8) and interleukin-6 (IL-6). IL-8 is a multifunctional cytokine, acting primarily as a neutrophil chemoattractant, while IL-6 is associated with poor metabolism in COPD patients. Since both cytokines play an important role in many lung diseases, such as COPD, pulmonary fibrosis or asthma; It seems reasonable to investigate the effect of novel cigarette filters on the levels of these cytokines in our recently developed complex lung model system. Inflammatory processes in the lung are associated with the production of various cytokines and the recruitment of neutrophils into the airways. IL-6 and IL-8 play crucial roles in the initiation and extension of inflammatory reactions. Exposure to cigarette smoke can activate inflammation by enhancing the secretion of pro-inflammatory cytokines, leading to chronic inflammation. Cigarette smoke can also cause organ disorders, such as destruction of the airways and loss of gas exchange surfaces, which can lead to impaired lung functions. All of these negative effects can contribute to the development of serious diseases, including COPD or cancer. Using 3D tissue culture as a test method, a combination of cells acting as a functional tissue unit can be evaluated in comparison to individual cells. The lung tissue comprises epithelial cells that have a distinguished cellular architecture. These cells have specialized cell-cell contacts, a polarized morphology, and bind to an underlying basement membrane. Maintaining these characteristics is essential for normal tissue function, which includes proliferation, differentiation, survival, and secretion. Cells grow naturally in a 3D environment. The spatial arrangement of cells within this environment affects how they interact with each other and their microenvironment. In turn, these intracellular signals affect morphology and a range of cellular functions. Therefore, when testing drug or toxic agent candidates using cell-based assays, the culture methods used should simulate the most natural representative in vivo form possible. The most natural cell growth method that simulates tissues for drug discovery applications is possibly 3D. In vitro tests for cigarette smoke are complicated. A large number of cell lines have been tested, but all have their own limitations. IL-8 and IL-6 can be produced by various inflammatory and lung cells, but investigation of a particular cell type can misrepresent the overall impact of smoke exposure. Cells grown in two-dimensional cell cultures are commonly used in various types of pharmacological tests, but these in vitro circumstances are less relevant to the in vivo situation than in the case of a three-dimensional model system. Three-dimensional lung cell cultures are more representative of what occurs in vivo, as they have an architecture and expression pattern that closely match the human lung. As the lung is a complex organ, it is necessary to investigate biological processes in a complex model system, since the arrangement of cells can affect the response given to a particular stimulus. Humeltis 3D lung tissue combines multiple cell types, representing the major cells of the airway tract.

Methods

Normal major human small airway epithelial cells (SAEC) and normal human lung fibroblasts (NHLF) were obtained from Lonza. These cells were isolated from anonymous donors of different sexes and ages. Human peripheral monocytes were isolated using the CD14 positive MicroBead isolation kit (Miltenyi Biotec). For 3D culture SAEC and NHLF cells were mixed in a 1: 1 ratio (SN spheroids), and human monocytes were also mixed with these human main cells (SNM spheroids). Cells were seeded in a low binding 96-well U-bottom plate. The spheroids were treated with cigarette smoke extracts (CSE) for 48 hours before measurement. The cigarettes used in the experiment were the Kentucky 3R4F Reference Cigarettes manufactured and assembled by the University of Kentucky, KY, USA The cigarettes were provided to OptiFilter Zrt of Hungary by Celanese Corporation, Narrows, VA, EE The cigarette filters were assembled, and the test cigarettes were produced by OptiFilter Zrt. CellFx filter rods were prepared and provided by Celanese Corporation. Celanese Corporation manufactured and provided additional acetate filter materials with different textural characteristics, thus producing different pressure drop values. The 27 mm acetate portions of the Kentucky Reference Cigarette (KRC) 3R4F filter (2.9 / 41,000) were removed and discarded. The filter rods, which are manufactured using Celanese's CellFx technology and containing different fillers, were inserted so that they face the burning surface of the cigarette. An additional part of acetate was selected and introduced into the filter, which ensures that the pressure drop value (resistance to total aspiration) of the cigarette (filter ventilation closed) was the same as the pressure drop value of the KRC (resistance to aspiration 170 mm H2O / - 2%). The Celanese rods were 12mm long. The acetate parts were 15mm long. The total length of the filter was 27 mm. A total of two different filters were manufactured, and the KRCs were fitted with them. Smoke from cigarettes that were fitted with filters of the present invention, CellFx filters containing different fillers, was measured and compared with a control in biological evaluation. The cigarette categorization was as follows:

Cigarette 1: Kentucky KRC Reference Cigarettes

Cigarette 2: CelRod-12-C Carbon Mono Rod Filter

Cigarette 3: Alginite Filter / CelRod-12-AG Grape Rod

CSE was prepared by bubbling the smoke of 2 cigarettes through 10 ml of cell culture medium at a constant air flow supplied by a Hydrotech vacuum pump (BioRad) for a total period of two minutes. The exposed medium was filtered under sterile conditions with a 0.22 µm syringe filter. The light scattering of the dissolved particles did not show significant differences within the 320-350 nm ranges. This solution was considered to be 100% E. CSE was prepared within 30 minutes for each experiment. The CSE (0.5%) was applied to the three-dimensional tissue cultures for 48 hours. After 48 hours the inflammatory cytokines produced by the 3D micro-tissues were measured in the supernatant medium using the BD Cytometric Bead Array Human Inflammatory Cytokine Kit (BD Biosciences). This kit provides a quantitative measurement of the level of IL-8 and IL-6 protein in tissue culture supernatant. The method relies on conjugated fluorescent microbeads of known size and a detection reagent, which provides a signal proportional to the amount of cytokine that binds. During 3 hours of incubation, the capture microbeads form a complex with the cytokine of the supernatant together with the detection reagent. Fluorescence intensity was analyzed with a FACS Canto II flow cytometer (BD Immunocytometry Systems, Erembodegem, Belgium) with the BD FACS DIVA V6 program, and the data were analyzed with the FCS Express V3 program. The Results represent the mean fluorescence intensity of the conjugated microbeads followed by the binding of IL-6 and IL-8.

Results

To investigate the production of inflammatory cytokines as a function of filter type, the spheroids were treated with CSE from standard cigarettes and two cigarettes containing different filters for 48 hours. The data shows that both IL-8 and IL-6 were reduced in macrophage-containing aggregates followed by CSE treatment of No. 3 filter cigarettes, indicating a reduced ability to initiate an inflammatory reaction. The differences were shown to be statistically significant for both cytokines.

Figure 14 shows human IL-8 protein in macrophage supernatants containing lung spheroids after 48 hours in 3 cell-like aggregates (SAEC, fibroblasts, and macrophages).

Figure 15 shows human IL-6 protein in macrophage supernatants containing lung spheroids after 48 hours.

The decrease in cytokine levels was statistically significant only in aggregates containing macrophages and only after 48 hours. In the aggregates that are formed only by fibroblasts and main epithelial cells (without macrophages), the reduction in cytokine levels was not significant either at 24 or 48 hours. Cigarette number 3 decreased the levels of both cytokines to the level that was determined in the control medium.

Conclusions

IL-6 and IL-8 play crucial roles in the initiation and propagation of inflammatory reactions. Exposure to cigarette smoke can activate inflammation by causing tissue damage, which enhances the secretion of pro-inflammatory cytokines, which can lead to chronic inflammation. Possibly, 3D human tissue cultures show a strong resemblance to the biochemical and pathological processes of human tissues in vivo. In this regard, it may be reasonable to assume that the statistically significant reduction in cytokines that were investigated in the immunologically active aggregates (containing macrophages), when smoke is filtered through filter number 3 could also be beneficial in the in vivo environment. . The data are summarized as follows (SN means aggregates containing major epithelial cells and fibroblasts, while SNM means aggregates containing epithelial cells, fibroblasts, and macrophages):

Summary

The above examples clearly demonstrate that alginite is especially effective when used in cigarette filters alone or in combination with other known components as discussed above. As unexpected and novel features of the invention, the use of alginite in cigarette filters resulted in significantly less reactive oxygen species (ROS) from saliva, significantly less ROS formation from blood serum, less endothelial damage, less lung epithelium damage, significantly higher glutathione level, less lung tissue damage, and less lung tissue inflammation.

In Vitro Biological Tests The biological tests that were selected were well chosen because they have a clear and well-established link to the in vivo biological pathways that have been documented to cause major smoking-related diseases. Additionally, in all cases the filters of the invention were shown to produce gas phase smoke which was much less damaging to cells in vitro tests than the smoke produced by the Kentucky reference filter. These are the results provide compelling evidence that cigarettes that were fitted with these filters can lessen the current health effects of smoking.

The examples further illustrated that alginite, even alone, has significantly better filtration characteristics over known filtration materials, and that alginite and prior art filtration materials act synergistically. Therefore, although not all the combinations they contain are mentioned in the examples, it is apparent to a person skilled in the art that any combination of alginite and filter materials known in the particular technical field will have the same properties.

Claims (17)

1. The use of alginite for filtering cigarette smoke. 2. The use according to claim 1 wherein the alginite is used in cigarette filters 3. The use of alginite for the preparation of cigarette filters 4. The use of claim 2 wherein the alginite is used alone or in combination with other substances that are used in cigarette filters to reduce the harmful effects of cigarette smoke. 5. The use of claim 4 wherein the other substances are selected from the group consisting of activated carbon or grape components. 6. The use of claim 5 wherein the other substance is activated carbon. 7. The use of claim 5 wherein the other substance is a component or are components of the grape. 8. The use according to claim 7 wherein the grape components are in the form of grape seed and skin grind. 9. Alginite for use in reducing the risk of harm from cigarette smoke in humans. 10. The alginite for the use of claim 9 wherein the alginite is used in the form of cigarette filters containing alginite. Alginite for use according to claim 9 wherein reducing the risk of damage means less ROS from saliva. 12. Alginite for use according to claim 9 wherein reducing the risk of damage means less ROS from serum. 13. Alginite for use according to claim 9 wherein reducing the risk of damage means less damage to the epithelial cells. 14. Alginite for use according to claim 9 wherein reducing the risk of damage means less damage to endothelial cells. 15. Alginite for use according to claim 9 wherein reducing the risk of damage means a higher glutathione level. 16. The alginite for the use according to claim 9 wherein reducing the risk of damage means less damage to the lung tissues. 17. Alginite for use according to claim 9 wherein reducing the risk of damage means less inflammation in the lung tissues.